Project Summary The overall goal of this proposal is to investigate the contribution of obesity to breast cancer initiation and progression by establishing the contribution of the insulin receptor (IR) signaling pathway to the regulation of breast cancer stem cells (CSCs). Obesity is a growing health epidemic that is associated with increased risk of both developing and dying from breast cancer. High insulin levels, which are a consequence of obesity, are an independent risk factor for breast cancer initiation and recurrence, which suggests a role for this signaling pathway in the regulation of breast CSCs. CSCs represent a sub-population of tumor cells that have the ability to self-renew and generate tumor heterogeneity and they are sufficient to initiate primary and recurrent tumor growth. Novel approaches are needed to target this aggressive tumor cell subpopulation. Although the involvement of the IR in regulating CSC function has been investigated in some cancer contexts, a rigorous analysis of the mechanism by which this receptor regulates CSC function has not been performed. This is clinically important because directly targeting the IR for cancer therapy is challenging given the essential role that this receptor plays in regulating normal metabolic homeostasis. The insulin receptor substrate (IRS) cytoplasmic adaptor proteins play key roles in the functional outcomes of IR signaling. In this proposal, the applicant advances the hypothesis that IRS2 mediates the regulation of breast CSCs by the IR, a function that cannot be executed by IRS1. The applicant?s preliminary data establish a role for IRS2 in the insulin-dependent regulation of CSC self-renewal and they identify a domain within IRS2 that is essential for this regulation. The ability of IRS2 to promote self-renewal also depends upon its recruitment and activation of PI3K. The applicant proposes that selectively disrupting functions of IRS2 that promote CSC self-renewal without interfering with its functions in normal metabolic homeostasis would be a novel approach for inhibiting IR regulation of CSCs. The results obtained from the experiments outlined in this proposal will lay the groundwork for developing targeted approaches that could be used in combination with current therapies to target CSCs to treat primary and recurrent breast tumors. To investigate the hypothesis that IRS2-dependent signaling in response to IR activation enhances CSC self-renewal the applicant will: 1) Investigate insulin-dependent regulation of breast CSCs. The hypothesis that insulin signaling through IR-A/IRS2 regulates breast CSC self-renewal will be examined; 2) Determine the mechanism by which insulin signaling regulates breast CSC function. The hypothesis that IR signaling through IRS2 enhances CSC self-renewal through the recruitment of factor(s) to a unique region within the C-terminal tail of IRS2 (SR) that cooperate with PI3K/AKT signaling will be examined; 3) Elucidate the mechanism by which hyperinsulinemia promotes breast tumor growth. The hypothesis that IR/IRS2 regulation of CSCs is required for enhancing tumor growth in response to hyperinsulinemic conditions will be examined using both genetic and drug resistance models.